Project Summary Pore-forming membrane channels are central mediators of many complex biological phenomena; such as synchronizing the contraction of our heart and electro-chemical signals in our brain, and detecting light, sound, touch, taste and smells of the world around us. This ability is dependent upon dynamic mechanism used to spatially and temporally modulate their cellular activity. Our research group is focused on understanding how these types of phenomena are choreographed by remarkably complex strategies of cell-to-cell communication, through the gap junctions. We aim to develop a molecular and atomic-level of mechanistic understanding of how gap junctions coordinate inter-cellular communication. To achieve this level of detail, we are combining the unique power of electron cryo-microscopy (CryoEM), together with targeted biophysical and functional studies to address several fundamental questions, such as: i) How do the gap junctions selectively control the flow of chemical information between cells? ii) How are their activities allosterically modulated by physiological cues? iii) How are cell-signaling platforms used to effectively control channel activity in a native multi-cellular environment? Despite their physiological and medical relevance, membrane proteins still only represent ~4% of the protein structure database. However, recent advances in the field of high-resolution single particle CryoEM, coupled with advancements in membrane protein biochemistry, are beginning to revolutionize the way we structurally characterize these proteins. With these technological tools in hand, we are addressing several key questions about gap junction selectivity and regulation. The results of our investigations are expected to provide an architectural framework and the mechanistic knowledge required for the development of targeted therapies against a range of gap junction related diseases, such as blindness, deafness, arrhythmia, stroke and cancers.!